Injuries to the anterior cruciate ligament (ACL) in the knee are common, with over 100,000 ACL reconstructions performed per year. There is currently no consensus on the ideal post-operative rehabilitation following ligament reconstruction. Knee ligament reconstruction procedures, such as for a ruptured anterior cruciate ligament (ACL), require transplantation of a tendon graft into bone tunnels. The tendon-to-bone healing process is not well understood. The long-term objective of this research is to understand the cellular and molecular events that control healing at the tendon-to-bone attachment site, and to understand the effect of mechanical load and inflammation on the healing process. Our central hypothesis is that mechanical load causing relative tendon-bone interface motion will improve healing only if it is applied after an initial period of immobilization to allow resolution of acute inflammation and initiation of early healing. We will use a rat model of ACL reconstruction in which a tendon graft is transplanted into bone tunnels in the femur and tibia. Graft-tunnel motion will be controlled using a specially-designed motorized loading device which will be used to apply 0, 1 or 10% axial cyclic strain to the healing bone-tendon-bone graft complex. Strain application will begin at 0, 7 or 14 days after surgery. Control animals (0% strain) will remain immobilized. We will evaluate healing at the tendon-bone interface at various time points after repair using histomorphometric, immunohistochemical, micro-CT, and biomechanical analyses of the bone-tendon-bone complex. We will examine the mechanism(s) by which mechanical stimulus affects healing by testing the hypothesis that early graft-tunnel motion results in increased macrophage accumulation, leading to TGF-beta production and excessive scar formation at the healing interface (rather than reformation of a normal ligament attachment site), while improved healing with delayed mobilization is associated with diminished macrophage activity. We will also evaluate healing in animals that are depleted of macrophages. These studies will provide important insight-into the relationship between mechanical load and inflammation on tendon-to-bone healing. The results of our studies will allow a scientific basis for the design of post- operative rehabilitation prescription.